線上固相微萃取液相層析質譜儀分析技術發展及酵母菌催化酮類立體選擇性反應之掌性二級醇產物分析

The development of on-line SPME-LC-MS analytical technique for chiral secondary alcohol product analysis with yeast mediated stereoselective reduction of ketones

粘祐銓(Yu-Chuan Nian)

指導教授 ： 鄭建業

中原大學/理學院/化學研究所/碩士(2013年)

https://doi.org/10.6840/cycu201301009

摘要

本研究針對樣品中微量物質，發展一種固相微萃取線上連結液相層析質譜儀的分析方法，以測定酵母菌立體選擇性催化4-苯基-2-丁酮還原反應之掌性二級醇R-及S-4-苯基-2-丁醇產物。萃取管柱使用思必可- Q多孔塗層中空開放毛細管柱，萃取動相、萃取流速及萃取時間分別為純水、每分鐘0.1毫升和0.95分鐘；反應物酮及掌性二級醇產物脫附的分離分析管柱為費洛美掌性OD管柱，脫附及分離分析的動相與流速分別為水/甲醇/異丙醇(體積比為55/25/20)及每分鐘0.5毫升。反應物酮及掌性二級醇均以質譜儀電噴灑離子化正離子模式偵測，偵測離子峰訊號為[M + Na]+。定量分析使用基質匹配外部標準品檢量線，線性範圍為0-50 μg mL-1，其線性迴歸係數為0.9950至0.9961之間，反應物酮與R-及S-二級醇產物之偵測極限值分別為0.02、0.01及0.01 μg mL-1，又線上固相微萃取連結液相層析質譜儀偵測的靈敏度，比液液萃取結合液相層析搭配紫外光偵測器的靈敏度要好25-40倍。
添加甘油的細胞反應介質使用於酵母菌催化還原4-苯基-2-丁酮模型化合物，生成 R-或S-4-苯基-2-丁醇，發現介質添加甘油的比例可影響產物的鏡像體選擇性。當反應介質的甘油/菌液體積比為10/90時，於受質濃度10 μg mL-1、25℃、pH 10.0、150 rpm轉速及不通入空氣下，有最佳之R-鏡相體過剩值(73.2%)；而甘油/菌液的體積比為30/70時，於受質濃度10 μg mL-1、34℃、pH 7.0、150 rpm轉速及不通入空氣的條件下，有最佳之S-鏡相體過剩值(87.1%)。細胞反應液經標準品添加測試之回收率，顯示分析的準確度介於84-91%，而分析的同日間與異日間之精確度，由重複分析量測的相對標準偏差值顯示分別介於10.8-21.1%及11.6-18.7%。

關鍵字

酵母菌 ； 釀酒酵母 ； 4-苯基-2-丁酮 ； R- 及 S-4-苯基-2-丁醇 ； 甘油/菌液均勻相溶液 ； 批次式反應 ； 掌性液相層析管柱 ； 多孔塗層中空開放毛細管柱 ； 線上相微萃取連結液相層析質譜儀 ； 液液萃取結合液相層析儀

並列摘要

In this study, the development of an on-line solid phase microextraction coupled liquid chromatography mass spectrometry (SPME-LC-MS) analytical method is aimed at the analysis of trace meterial in a sample of Saccharomyces cerevisiae mediated stereoselective reduction of a model compound 4-phenyl-2-butanone to chiral secondary alcohol products R- and S-4-phenyl-2-butanol. The extraction column was Supel-Q PLOT capillary column with water as the extraction mobile phaes at an extraction flow rate of 0.1 mL min-1 and the extraction time was 0.95 min. The analytical separation column used for the desorbed ketone and chiral secondary alcohols was phenomenex Lux 3u Cellulose-1 Chiral OD with the desorption and analysis mobile phase of water/methanol/isopropanol (55/25/20, v/v/v) and a flow rate of 0.5 mL min-1. The detection of ketone and chiral secondary alcohols with mass spectrometry was electrospray ionization in positive ion mode and the detection signal was [M + Na]+. The quantitative analysis was by matrix-matched external standard calibration method, the linear range of the standard calibration curve was 0 to 50 μg mL-1 with the linear regression coefficient in the range 0.9950 to 0.9961. The limit of detection (LOD) were 0.02, 0.01 and 0.01μg mL-1, respectively. In addition, the sensitivity of on-line SPME-LC-MS for ketone and R- and S-chiral secondary alcohol products was 25 to 40 folds better than that of liquid-liquid extraction (LLE) coupled high-performance liquid chromatography ultraviolet detection (HPLC-UV).
As 4-phenyl-2-butanone has been used as a model substrate for the S. cerevisiae mediated stereoselective reduction to the R- or S-4-phenyl-2-butanol product, the addition of glycerol in different volume ratio to the reaction cell culture affects the enantioselectivity of yeast. The batch fermentation experiment using a reaction medium glycerol/aqueous cell culture (10/90, v/v), a substrate concentration of 10 μg mL-1, a temperature of 25℃, a pH 10.0, a stirring speed of 150 rpm, and without air bubbling, demonstrated that the reaction product is in excess of R-4-phenyl-2-butanol with the enantiomeric excess (e.e.) value of 73.2%. The batch fermentation experiment using a reaction medium glycerol/aqueous cell culture (30/70, v/v), a substrate concentration of 10 μg mL-1, a temperature of 34℃, a pH 7.0, a stirring speed of 150 rpm, and without air bubbling, demonstrated that the reaction product is in excess of S-4-phenyl-2-butanol with the e.e. value of 87.5%. The analysis accuravy by the spiking experiments of the cell culture were 84-91%. The intra-day precision and the inter-day precision by calculating the relative standard deviations (RSDs) of repeated measurements were in the range from 10.8-21.1% and 11.6-18.7%, respectively.

並列關鍵字

S-4-phenyl-2-butanol ； glycerol/yeast culture homogeneous solution ； R-4-phenyl-2-butanol ； 4-phenyl-2-butanone ； Saccharomyces cerevisiae ； Yeast ； Chiralcel liquid chromatographic column ； batch reaction ； PLOT capillary column ； liquid-liquid extraction combined high-performance liquid chromatography with ultraviolet detector ； on-line solid-phase microextraction coupled liquid chromatography mass spectrometry

參考文獻

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